The role of kinetochore dynein in checkpoint silencing is restricted to disassembly of the corona.

During mitosis, kinetochore-microtubule attachments are monitored by a molecular surveillance system known as the spindle assembly checkpoint. The prevailing model posits that dynein evicts checkpoint proteins (e.g.

Production and purification of recombinant monoclonal antibodies from human cells based on a primary sequence.

There are challenges to using commercially available antibodies generated in animals, including concerns with reproducibility, high costs, and ethical issues. Here, we present a protocol for generating and purifying recombinant antibodies from human HEK293 suspension culture cells from a primary sequence. We describe the steps to generate antibody heavy and light chain plasmids, followed by transfection of the plasmids into cells and purification of antibodies.

Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities.

Aneuploidy, the incorrect number of whole chromosomes, is a common feature of tumors that contributes to their initiation and evolution. Preventing aneuploidy requires properly functioning kinetochores, which are large protein complexes assembled on centromeric DNA that link mitotic chromosomes to dynamic spindle microtubules and facilitate chromosome segregation. The kinetochore leverages at least two mechanisms to prevent aneuploidy: error correction and the spindle assembly checkpoint (SAC).

Generation and diversification of recombinant monoclonal antibodies.

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences.

The Hec1/Ndc80 tail domain is required for force generation at kinetochores, but is dispensable for kinetochore-microtubule attachment formation and Ska complex recruitment.

The conserved kinetochore-associated NDC80 complex (composed of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including 1) connecting mitotic chromosomes to spindle microtubules to establish force-transducing kinetochore-microtubule attachments and 2) regulating the binding strength between kinetochores and microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to microtubules, additional factors support these processes as well, including the spindle and kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind microtubules, especially to depolymerizing microtubule plus ends, but how this is accomplished remains unclear.

Aurora B kinase is recruited to multiple discrete kinetochore and centromere regions in human cells.

Aurora B kinase has a critical role in regulating attachments between kinetochores and spindle microtubules during mitosis. Early in mitosis, kinase activity at kinetochores is high to promote attachment turnover, and in later mitosis, activity decreases to ensure attachment stabilization. Aurora B localizes prominently to inner centromeres, and a population of the kinase is also detected at kinetochores.

Use of Confocal Microscopy to Evaluate Equine Zygote Development After Sperm Injection of Oocytes Matured In Vivo or In Vitro.

Confocal microscopy was used to image stages of equine zygote development, at timed intervals, after intracytoplasmic sperm injection (ICSI) of oocytes that were matured in vivo or in vitro. After fixation for 4, 6, 8, 12, or 16 h after ICSI, zygotes were incubated with α/β tubulin antibodies and human anticentromere antibody (CREST/ACA), washed, incubated in secondary antibodies, conjugated to either Alexa 488 or Alexa 647, and incubated with 561-Phalloidin and Hoechst 33258. An Olympus IX81 spinning disk confocal microscope was used for imaging.

Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore-microtubule dynamics.

Precise regulation of kinetochore-microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates kinetochore substrates to promote microtubule turnover. A critical target of Aurora B is the N-terminal "tail" domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between kinetochores and microtubules.

Real-time quantification of single RNA translation dynamics in living cells.

Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time in vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds.

Superresolved multiphoton microscopy with spatial frequency-modulated imaging.

Superresolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all superresolution imaging techniques reported to date rely on real energy states of fluorescent molecules to circumvent the diffraction limit, preventing superresolved imaging with contrast mechanisms that occur via virtual energy states, including harmonic generation (HG). We report a superresolution technique based on spatial frequency-modulated imaging (SPIFI) that permits superresolved nonlinear microscopy with any contrast mechanism and with single-pixel detection.

Measuring Kinetochore-Microtubule Attachment Stability in Cultured Cells.

Duplicated sister chromatids connect to the mitotic spindle through kinetochores, large proteinaceous structures built at sites of centromeric heterochromatin. Kinetochores are responsible for harnessing the forces generated by microtubule polymerization and depolymerization to power chromosome movements. The fidelity of chromosome segregation relies on proper kinetochore function, as precise regulation of the attachment between kinetochores and microtubules is essential to prevent mitotic errors, which are linked to the initiation and progression of cancer and the formation of birth defects (Godek et al.

Cytoskeletal alterations associated with donor age and culture interval for equine oocytes and potential zygotes that failed to cleave after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an established method to fertilise equine oocytes, but not all oocytes cleave after ICSI. The aims of the present study were to examine cytoskeleton patterns in oocytes after aging in vitro for 0, 24 or 48h (Experiment 1) and in potential zygotes that failed to cleave after ICSI of oocytes from donors of different ages (Experiment 2). Cytoplasmic multiasters were observed after oocyte aging for 48h (P<0.

Accurate phosphoregulation of kinetochore-microtubule affinity requires unconstrained molecular interactions.

Accurate chromosome segregation relies on dynamic interactions between microtubules (MTs) and the NDC80 complex, a major kinetochore MT-binding component. Phosphorylation at multiple residues of its Hec1 subunit may tune kinetochore-MT binding affinity for diverse mitotic functions, but molecular details of such phosphoregulation remain elusive. Using quantitative analyses of mitotic progression in mammalian cells, we show that Hec1 phosphorylation provides graded control of kinetochore-MT affinity.

KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting Aurora B kinase activity.

Aurora B kinase phosphorylates kinetochore proteins during early mitosis, increasing kinetochore–microtubule (MT) turnover and preventing premature stabilization of kinetochore–MT attachments. Phosphorylation of kinetochore proteins during late mitosis is low, promoting attachment stabilization, which is required for anaphase onset. The kinetochore protein KNL1 recruits Aurora B–counteracting phosphatases and the Aurora B–targeting factor Bub1, yet the consequences of KNL1 depletion on Aurora B phospho-regulation remain unknown.

Super-resolution photon-efficient imaging by nanometric double-helix point spread function localization of emitters (SPINDLE).

Super-resolution imaging with photo-activatable or photo-switchable probes is a promising tool in biological applications to reveal previously unresolved intra-cellular details with visible light. This field benefits from developments in the areas of molecular probes, optical systems, and computational post-processing of the data. The joint design of optics and reconstruction processes using double-helix point spread functions (DH-PSF) provides high resolution three-dimensional (3D) imaging over a long depth-of-field.

Temporal changes in Hec1 phosphorylation control kinetochore-microtubule attachment stability during mitosis.

Precise control of the attachment strength between kinetochores and spindle microtubules is essential to preserve genomic stability. Aurora B kinase has been implicated in regulating the stability of kinetochore-microtubule attachments but its relevant kinetochore targets in cells remain unclear. Here, we identify multiple serine residues within the N-terminus of the kinetochore protein Hec1 that are phosphorylated in an Aurora-B-kinase-dependent manner during mitosis.